Structure-activity relationships of pyrroloquinazolines as thrombin receptor antagonists.

A series of pyrroloquinazolines has been discovered that represent novel small molecule inhibitors of the intramolecular ligand of the thrombin receptor. Analogs were prepared to study the structure-activity relationships of substitution at the N 1, N3, and N7 positions of the heterocycle. Compounds 4e and 4f have been identified with IC50's of 56 and 52 nM, respectively.

Binding of a thrombin receptor tethered ligand analogue to human platelet thrombin receptor.

A thrombin receptor-radioligand binding assay was developed using [3H]A(pF-F)R(ChA)(hR)Y-NH2 ([3H]haTRAP), a high affinity thrombin receptor-activating peptide (TRAP), and human platelet membranes. Scatchard analysis of saturation binding data indicated that [3H]haTRAP bound to platelet membranes with a Kd of 15 nM and a Bmax of 5.2 pmol/mg of protein. The binding was reduced by GPPNHP, a nonmetabolizable GTP analogue. Various TRAPs and a TRAP antagonist, but not other receptor agonists, displaced [3H]haTRAP from the binding sites. SFLLRN-NH2, a thrombin receptor-tethered ligand analogue, and [3H]haTRAP exhibited competitive binding for the same binding sites. The relative affinity of these peptides for the binding site paralleled their EC50 or IC50 values for platelet aggregation. These data indicate that [3H]haTRAP binds specifically and saturably to the functioning G protein-linked thrombin (tethered ligand) receptor in human platelet membranes.

Cyclic nucleotide phosphodiesterase isozymes in rat mesangial cells.

We characterized cyclic nucleotide phosphodiesterases isolated from rat mesangial cells and assessed their roles in regulating cellular cyclic nucleotide levels. Three peaks of phosphodiesterase activity were eluted by a linear sodium acetate gradient from a Q Sepharose column loaded with the mesangial cell extract. The first peak activity was stimulated by Ca(2+)-calmodulin and inhibited by calmodulin-stimulated phosphodiesterase inhibitors but not by a selective cGMP specific phosphodiesterase V inhibitor. The second, minor activity peak was stimulated by cyclic GMP and inhibited by EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], a selective inhibitor of cyclic GMP-stimulated phosphodiesterase II. The last peak activity was not inhibited by cyclic GMP but selectively inhibited by rolipram [4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidene] or Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone], inhibitors of cyclic AMP specific, cyclic GMP insensitive phosphodiesterase IV. Based on their order of chromatographic elution, kinetic properties and sensitivity to allosteric agents and inhibitors, the peak 1, 2 and 3 correspond to phosphodiesterase I, II and IV. The basal cyclic GMP level was raised more effectively by selective inhibitor of phosphodiesterase I than phosphodiesterase II. In contrast, the atrial natriuretic factor-induced cyclic GMP elevation was potentiated more effectively by selective inhibitors of phosphodiesterase II than phosphodiesterase I. The forskolin-induced cyclic AMP increase was greatly potentiated by selective phosphodiesterase IV inhibitors but not by other phosphodiesterase inhibitors. These data suggest that phosphodiesterase I and II are responsible for cyclic GMP hydrolysis whereas phosphodiesterase IV is mainly responsible for cyclic AMP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid receptor-mediated catabolism of 125I-atrial natriuretic factor by vascular endothelial cells.

The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.

Metabolism of 125I-atrial natriuretic factor by vascular smooth muscle cells. Evidence for a peptidase that specifically removes the COOH-terminal tripeptide.

The addition of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) to cultured bovine aortic smooth muscle cells at 37 degrees C resulted in a rapid clearance from the medium (t1/2 approximately 7.5 min). Within 5 min, [125I]iodotyrosine126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg-[125]iodotyrosine126 (125I-FRY) appeared in the medium. The identities of these degradation products were confirmed by 1) retention time on high performance liquid chromatography (HPLC) relative to standards, 2) products generated by digestion with aminopeptidase M, and 3) the absence of the Met110. Preincubation of the cells with ammonium chloride or chloroquine resulted in a significant increase in the intracellular accumulation of radiolabel, indicative of endocytosis and rapid delivery of 125I-hANF to an acidic intracellular compartment (endosome and/or lysosome). Neither ammonium chloride, chloroquine, nor excess unlabeled hANF blocked the rapid appearance in the medium of 125I-RY or 125I-FRY. Bestatin inhibited the generation of 125I-RY, with a concomitant increase in 125I-FRY, suggesting that the 125I-RY is produced by aminopeptidase action on 125I-FRY. The endopeptidase 24.11 (enkephalinase) inhibitor, SCH 39370, did not inhibit the formation of 125I-FRY. These results provide evidence of a peptidase capable of specifically removing the COOH-terminal tripeptide from 125I-hANF. The COOH-terminal tripeptide, Phe124-Arg-Tyr126, was also isolated from cell digests of hANF by HPLC and its identity confirmed by amino acid analysis. Since it is generally believed that the COOH-terminal tripeptide is critical to many of atrial natriuretic factor-(99-126)'s bioactivities, this enzyme may be involved in the inactivation of atrial natriuretic factor-(99-126) in target tissues.